The long-term goal of our research is to elucidate, in molecular terms, the structure, function, and extracellular metabolism of human high density lipoprotein (HDL) subfractions. In this project we focus on the regulation of lecithin cholesterol acyltransferase (LCAT), the enzyme that carries out the esterification of cholesterol on HDL, and is involved in the removal of cholesterol from cells and from more buoyant lipoproteins, and in the transformations of HDL subspecies in plasma. Our objectives are to elucidate the interfacial factors that control the interactions of LCAT with the surface of lipoproteins, and the mechanism for the activation of the enzymatic reaction on HDL We propose to study the binding and steady state kinetics of pure LCAT with reconstituted HDL (rHDL), having precisely defined interfacial properties, and with narrow subfractions of native HDL, separated according to their apolipoprotein composition and size. The binding affinities will be determined by a new, activity - inhibition method, and by observing the changes in fluorescence properties of fluorescently labeled LCAT upon binding to interfaces. The kinetic measurements, in conjunction with the dissociation constants, will be used to obtain the intrinsic kinetic parameters for the reaction of LCAT with its substrate lipids. The mechanism for the activation of the LCAT reaction on HDL will be explored by studying the effect of apolipoprotein A-I on the binding of LCAT to lipid interfaces, the proximity of LCAT to the apolipoprotein, the conformational changes in LCAT upon binding to interfaces, and possible specific interactions of the apolipoprotein with lipids. We expect the results of these studies to define the interfacial properties of HDL subspecies which determine the binding and activity of LCAT, and to provide a firm experimental basis in formulating a hypothesis for the activation of the LCAT reaction on HDL.